MOPS Buffer Research Articles

Preparation, characterization, and bioavailability of a phosphorylated human‐like collagen calcium complex

The aim of this study was to determine whether a phosphorylated human‐like collagen calcium (PHLC–Ca) complex showed a higher bioavailability than other calcium supplements. To improve the calcium‐binding ability of human‐like collagen (HLC), modified HLC was prepared by introducing a reactive phosphate group using a dry‐heating method, followed by the formation of PHLC–Ca in MOPS buffer. PHLC–Ca was characterized using atomic absorption spectrophotometry, Fourier transform infrared (FTIR), fluorescence quenching, circular dichroism (CD), and ultraviolet–visible absorption (UV–vis) spectroscopy. The calcium uptake test showed that the bioavailability of PHLC–Ca was higher than that of CaCl2 and G‐Ca. Data from a mouse model of osteoporosis suggested that PHLC–Ca is superior for treating osteoporosis than CaCl2 or G‐Ca. These results indicate that PHLC–Ca is a desirable calcium supplement for use in daily life and clinical application. Copyright © 2015 John Wiley & Sons, Ltd.

MOPS Buffer (10×)

MOPS Buffer (10×)

An “off–on” optical sensor for mercury ion detection in aqueous solution and living cells

A rhodamine-based optical sensor Rh–3S has been developed for selective detection of Hg2+ in aqueous solution as well as in living cells. UV–vis and fluorescence spectroscopic studies reveal that Rh–3S shows marked sensitivity and selectivity to Hg2+ with a chelation-induced “off–on” response in acetonitrile (ACN)/MOPS buffer (10mM, pH7.3, v/v, 1:1). The Rh–3S sensor binds Hg2+ in a 1:1 stoichiometry with an apparent binding constant 3.71×106M−1 (log K=6.57) and displays a distinct change in color and fluorescence upon the alteration of free Hg2+ levels in solution with a reversible response and little interference with other biological relevant metal ions. Live cell confocal imaging studies demonstrate that the sensor is also capable of imaging the presence of Hg2+ ions as well as its dynamic changes in live cells.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

DNA-Based Asymmetric Catalysis: Role of Ionic Solvents and Glymes.

Recently, DNA has been evaluated as a chiral scaffold for metal complexes to construct so called 'DNA-based hybrid catalysts', a robust and inexpensive alternative to enzymes. The unique chiral structure of DNA allows the hybrid catalysts to catalyze various asymmetric synthesis reactions. However, most current studies used aqueous buffers as solvents for these asymmetric reactions, where substrates/products are typically suspended in the solutions. The mass transfer limitation usually requires a long reaction time. To overcome this hurdle and to advance DNA-based asymmetric catalysis, we evaluated a series of ionic liquids (ILs), inorganic salts, deep eutectic solvents (DES), glymes, glycols, acetonitrile and methanol as co-solvents/additives for the DNA-based asymmetric Michael addition. In general, these additives induce indistinguishable changes to the DNA B-form duplex conformation as suggested by circular dichroism (CD) spectroscopy, but impose a significant influence on the catalytic efficiency of the DNA-based hybrid catalyst. Conventional organic solvents (e.g. acetonitrile and methanol) led to poor product yields and/or low enantioselectivities. Most ILs and inorganic salts cause the deactivation of the hybrid catalyst except 0.2 M [BMIM][CF3COO] (95.4% ee and 93% yield) and 0.2 M [BMIM]Cl (93.7% ee and 89% yield). Several other additives have also been found to improve the catalytic efficiency of the DNA-based hybrid catalyst (control reaction without additive gives >99% ee and 87% yield): 0.4 M glycerol (>99% ee and 96% yield at 5 °C or 96.2% ee and 83% yield at room temperature), 0.2 M choline chloride/glycerol (1:2) (92.4% ee and 90% yield at 5 °C or 94.0% ee and 88% yield at room temperature), and 0.5 M dipropylene glycol dimethyl ether (>99% ee and 87% yield at room temperature). The use of some co-solvents/additives allows the Michael addition to be performed at a higher temperature (e.g. room temperature vs 5 °C) and a shorter reaction time (24 h vs 3 days). In addition, we found that a brief pre-sonication (5 min) of DNA in MOPS buffer prior to the reaction could improve the performance of the DNA-based hybrid catalyst. We have also shown that this DNA-based catalysis method is suitable for a variety of different substrates and relatively large-scale reactions. In conclusion, a judicious selection of benign co-solvents/additives could improve the catalytic efficiency of DNA-based hybrid catalyst.

基于Con A门控分子的甘露糖响应型介孔二氧化硅纳米控制释放体系

In this work, a novel delivery system based on Concanavalon A(Con A)-gated glucose-functionalized mesoporous silica nanoparticle (MSN-Glc-Con A) was constructed for the mannose-responsive controlled release. In this system, glucose was firstly modified on the surface of mesoporous silica nanoparticles (MSN) through the crosslinking reaction between glucosamine and cyanato-functionalized MSN. Con A, a lectin, was subsequently bound the functionalized glucose epitopes through multivalent carbohydrate- protein interactions, resulting in blockage of pores and inhibition of guest molecules release. In the presence of mannose, the Con A competitively bound with mannose and got away from the pore of MSN because of higher affinity for mannose. Thus, the pores were uncapped and the entrapped guest molecules were released. The physical and chemical properties of the as-synthesized MSN were characterized by transmission electron microscopy, X-ray diffraction, Fourier transform Infrared spectroscopy, Brunauer-Emmett- Teller and thermogravimetric analysis. As a proof-of-principle, [Ru(bipy)3]Cl2 was selected as the model guest molecules, and the mannose-responsive loading and release of [Ru(bipy)3]Cl2 have been investigated. The results demonstrate that the system had excellent loading amount (89.90 ± 7.73 μmol/g SiO2) and good mannose-responsive release behavior. Release-profile studies in MOPS buffer showed that no [Ru(bipy)3]Cl2 leaked when the pore was closed and that release occurred immediately after adding mannose. The dye release percentage can over 90% after treatment with 40 mmol/L mannose for 340 min. This lectin-gated MSN delivery system is a promising candidate for therapeutic agent delivery.

Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum.

BackgroundCholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs, and are considered as potential antibacterial drug targets.ResultsThe gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten like kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent Km = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS.ConclusionCgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications.

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.

In vitro screening of forty medicinal plant extracts from the United States Northern Great Plains for anthelmintic activity against Haemonchus contortus

An egg hatch assay (EHA) and a larval migration assay (LMA) involving Haemonchus contortus was used to evaluate the anthelmintic activity of methanol extracts from 40 plants that are native or naturalized within the U.S.A. Northern Great Plains. Only one of these 40 plants (i.e. Lotus corniculatus) had been previously evaluated for activity against any gastrointestinal nematode. The various extracts were initially screened at 50mg/ml diluted either in 0.5% dimethyl sulphoxide (DMSO) or 3-(N-morpholino) propanesulfonic acid (MOPS buffer), and plants showing 100% inhibition at 50mg/ml, were further evaluated at 8 other concentrations (25–0.19mg/ml). Extracts with 100% activity with the EHA were again screened with the LMA (50mg/ml). Two extracts with the highest LMA inhibition were also evaluated at lower concentrations (25–3.1mg/ml). Of the 40 methanolic extracts screened, 7 (Chrysothamnus viscidiflorus, Ericameria nauseosa, Liatris punctata, Melilotus alba, Melilotus officinalis, Perideridia gairdneri, and Sanguinaria canadensis) showed significant egg-hatch inhibition in DMSO and MOPS buffer. Three extracts (Geranium viscosissimum, L. corniculatus, and Rhus aromatica) only showed significant inhibition in DMSO. The 8 extracts showing 100% efficacy at 50mg/ml exhibited dose-dependent effects at the 8 lower concentrations, and R. aromatica and E. nauseosa extracts had the lowest ED50 values. Similarly, when these 8 plant extracts were further evaluated with the LMA, the extracts of E. nauseosa and R. aromatica again exhibited the highest activity (p<0.001), with ED50 values of 4.0mg/ml and 10.43mg/ml respectively. Three other extracts (C. viscidiflorus, M. alba and M. officinalis) also showed inhibitory activity in the LMA. These results support the need for additional evaluations of the nematocidal properties for at least these 5 plants.

Development of a chemically defined medium for the production of the antibiotic platensimycin by Streptomyces platensis

The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.

Characterization of l-cysteine capped CdTe quantum dots and application to test Cu(II) deficiency in biological samples from critically ill patients

The catalytic activity of copper ion gives, from the physiological point of view, a central role in many biological processes. Variations in the composition and location of cellular copper have been addressed given their physiological and pathological consequences. In this paper l-cysteine capped CdTe quantum dots is used for the fluorimetric determination of Cu(II) in biological samples from healthy individuals and patients admitted to the Intensive Care Units (ICU). An acceptable homogeneity in the CdTe QDs size has been obtained with an average value of 3nm. No significant alterations in the spectral properties were observed for 2 months when stored in vacutainers at 6°C and a concentration of approximately 2μM. Data from oxidative stress markers such superoxide dismutase, total antioxidant capacity and DNA damage can be correlated with a Cu(II) deficiency for the ICU patients as measured by flame-atomic absorption spectroscopy (FAAS) and inductively coupled plasma source mass spectrometry (ICP-MS). Aqueous solutions 0.3μM of l-cysteine capped CdTe QDs in MOPS buffer (6mM, pH 7.4) used at 21°C in the range 15–60min after preparation of the sample for the measurements of fluorescence gives contents in Cu(II) for erythrocytes in good agreement with those obtained in FAAS and ICP-MS but the comparative ease of use makes the fluorimetric technique more suitable than the other two techniques for routine analysis.

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure–activity changes

The purpose of the present investigation was to study the pH dependence of both the immobilization process and the enzyme activity of a feruloyl esterase (FoFaeC from Fusarium oxysporum) immobilized in mesoporous silica. This was done by interpreting experimental results with theoretical molecular modeling of the enzyme structure. Modeling of the 3D structure of the enzyme together with calculations of the electrostatic surface potential showed that changes in the electrostatic potential of the protein surface were correlated with the pH dependence of the immobilization process. High immobilization yields were associated with an increase in pH. The transesterification activity of both immobilized and free enzyme was studied at different values of pH and the optimal pH of the immobilized enzyme was found to be one unit lower than that for the free enzyme. The surface charge distribution around the binding pocket was identified as being a crucial factor for the accessibility of the active site of the immobilized enzyme, indicating that the orientation of the enzyme inside the pores is pH dependent. Interestingly, it was observed that the immobilization pH affects the specific activity, irrespective of the changes in reaction pH. This was identified as a pH memory effect for the immobilized enzyme. On the other hand, a change in product selectivity of the immobilized enzyme was also observed when the transesterification reaction was run in MOPS buffer instead of citrate phosphate buffer. Molecular docking studies revealed that the MOPS buffer molecule can bind to the enzyme binding pocket, and can therefore be assumed to modulate the product selectivity of the immobilized enzyme toward transesterification.

Reply to Campos and Muñoz: Why phosphate is a bad buffer for guanidinium chloride titrations

Liu et al. (1) alleged that they could not repeat our work on the protein BBL (Protein Data Bank ID code 1W4H) at the most basic level. Campos and Munoz now admit (2), as we explained (3), that they used a phosphate buffer that greatly changed pH during the guanidinium chloride (GdmCl) titration relative to our Mops buffer. They can now repeat our work.

The preparation and characterization of novel human-like collagen metal chelates

In order to develop the nutritional trace elements which could be absorbed and utilized effectively, protein chelates were adopted. Calcium, copper and manganese were considered based on their physiological functions, and the new chelates of HLC-Ca, HLC-Cu and HLC-Mn were formed in MOPS or MES buffer and purified by gel chromatography, and then freeze-dried. And they were detected and analyzed by atomic absorption spectrophotometry, ultraviolet–visible absorption (UV–vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, fluorescence quenching method, circular dichroism (CD) and differential scanning calorimetry (DSC). The results showed that some chemical reactions happened between HLC and the three metal ions to form new chemical compounds. The thermodynamic parameters, ∆H, ∆G and ∆S, showed that the chelation process between HLC and metal ions was performed spontaneously. Fluorescence quenching spectra of HLC indicated that the quenching mechanism was static in nature. According to the data of DSC, the new chelates were more stable than the free HLC. And HLC-metal complex was non-toxic to the BHK21 cell through MTT assay.

Don’t waste good methods on bad buffers and ambiguous data

Recently in PNAS, Liu et al. (1) presented single-molecule fluorescence resonance energy transfer experiments (sm-FRET) on the protein BBL (QNND-BBL) that claim to show there is only one populated state during folding rather than the two separate states we had previously, clearly observed (2). Liu et al. (1) stated about our work: “… we were unable to reproduce their results even at the most basic level. For instance, the equilibrium chemical unfolding curve of QNND-BBL at 279 K has Cm of approximately 2.4 M GdmCl and not 4 M GdmCl as reported before …” That claim is untrue. Liu et al. did not repeat our experiments but replaced our Mops buffer by phosphate, which dramatically changes pH during the experiment to much lower values where the protein is far less stable. Liu et al. (1) did their GdmCl denaturation in supposedly pH 7 phosphate buffer. It is well known (3) that anionic buffers are sensitive to salt concentration—and dianionic phosphate exquisitely so—whereas amine buffers are rather insensitive. Accordingly, we always use and recommend (4) amine buffers for GdmCl titrations. We performed our denaturation experiments in a Mops buffer, the pH of which at 279 K is constant at 7.35 ± 0.05 from 0 to 6 M GdmCl (its pH at 298 K is 7). We found the pH of the phosphate buffer used by Liu et al. dropped rapidly to below pH 6 and was pH 5.5 at the denaturation midpoint of the transition in Mops buffer (Fig. 1A). They thus progressively lowered the pH during the denaturation transition by one to two units below that of ours. The stability of BBL drops rapidly with decreasing pH below 7 (Fig. 1B) (5), and so Liu et al. (1) found a lower artifactual midpoint (Cm). We actually reported one experiment in phosphate buffer at pH 5.8 at 2.5 M GdmCl (ref. 2 and figure S3 in ref. 2), which shows the protein to be largely denatured, unlike for the titrations in Mops buffer with Cm = 4 M (figure S1 in ref. 2), consistent with the Liu et al. data.

Determination of Zn-citrate in human milk by CIM monolithic chromatography with atomic and mass spectrometry detection

In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in cow's milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk since analytical procedures reported were not quantitative. So, complex investigation was carried out to develop analytical method for quantitative determination of this biologically important molecule. Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation of Zn-citrate complexes ([Zn(Cit)]− and [Zn(Cit)2]4−; column recoveries 94–102%) and good repeatability and reproducibility of results with relative standard deviation (RSD±3.0%) were obtained. In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01μg ZnmL−1 and 0.0005μg ZnmL−1, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical tool for investigation of the percentage and the amount of Zn-citrate in human milk.

A novel sucrose phosphorylase from the metagenomes of sucrose-rich environment: isolation and characterization

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12 mM, a V(max) of 69.24 μmol min(-1) mg(-1) and a k(cat) of 31.19 s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L-arabinose, D-fructose and L-sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L-arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9 %) in phosphate buffer was even higher than that (5.8 %) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.

Selective fluorimetric detection of cadmium in a microfluidic device

A microfluidic device equipped with a fluorimetric detection has been developed for the flow injection detection of cadmium, a very toxic metal. The fluorescent sensor, Rhod-5N, is a water-soluble commercial dye whose fluorescence is increased in presence of cadmium. A detection limit of 0.45μgL−1 has been determined in MOPS buffer at pH 7. Special attention was paid to the interference of other metal ions. The interference of Pb2+ was circumvented by solid phase adsorption (SPE) on aminopropyl silica. Langmuir analysis of the adsorption isotherms has shown a better affinity of this functionalized silica towards Pb2+. After optimization of both pH and flow rate of the analyte through the SPE minicolumn, cadmium could be determined even in presence of lead at a concentration 10 times larger.

Mercury/Homocysteine Ligation-Induced ON/OFF-Switching of a T–T Mismatch-Based Oligonucleotide Molecular Beacon

A molecular beacon (MB) with stem-loop (hairpin) DNA structure and with attached fluorophore-quencher pair at the ends of the strand has been applied to study the interactions of Hg(2+) ions with a thymine-thymine (T-T) mismatch in Watson-Crick base-pairs and the ligative disassembly of MB·Hg(2+) complex by Hg(2+) sequestration with small biomolecule ligands. In this work, a five base-pair stem with configuration 5'-GGTGG...CCTCC-3' for self-hybridization of MB has been utilized. In this configuration, the four GC base-pair binding energy is not sufficient to hybridize fully at intermediate temperatures and to form a hairpin MB conformation. The T-T mismatch built-in into the stem area can effectively bind Hg(2+) ions creating a bridge, T-Hg-T. We have found that the T-Hg-T bridge strongly enhances the ability of MB to hybridize, as evidenced by an unusually large MB melting temperature shift observed on bridge formation, ΔT(m) = +15.1 ± 0.5 °C, for 100 nM MB in MOPS buffer. The observed ΔT(m) is the largest of the ΔT(m) found for other MBs and dsDNA structures. By fitting the parameters of the proposed model of reversible MB interactions to the experimental data, we have determined the T-Hg-T bridge formation constant at 25 °C, K(1) = 8.92 ± 0.42 × 10(17) M(-1) from mercury(II) titration data and K(1) = 1.04 ± 0.51 × 10(18) M(-1) from the bridge disassembly data; ΔG° = -24.53 ± 0.13 kcal/mol. We have found that the biomarker of oxidative stress and cardiovascular disease, homocysteine (Hcys), can sequester Hg(2+) ions from the T-Hg-T complex and withdraw Hg(2+) ions from MB in the form of stable Hg(Hcys)(2)H(2) complexes. Both the model fitting and independent (1)H NMR results on the thymidine-Hg-Hcys system indicate also the high importance of 1:1 complexes. The high value of K(1) for T-Hg-T bridge formation enables analytical determinations of low concentrations of Hg(2+) (limit of detection LOD = 19 nM or 3.8 ppb, based on 3σ method) and Hcys (LOD = 23 nM, 3σ method). The conditional stability constants for Hg(Hcys)H(2)(2+) and Hg(Hcys)(2)H(2) at 52 °C have been determined, β(112) = 5.37 ± 0.3 × 10(46) M(-3), β(122) = 3.80 ± 0.6 × 10(68) M(-4), respectively.